Cell sheet construct for neurovascular reconstruction and manufacture thereof

ABSTRACT

The invention relates to a cell sheet construct for neurovascular reconstruction. The cell sheet construct has a vascular endothelial cell layer and a neural stem cell layer, and the two layers are physically in direct contact with each other, where the vascular endothelial cell layer forms branching vasculatures, and the neural stem cell layer differentiates into neurons. The invention also relates to a method for manufacturing the cell sheet construct, having the following steps: culturing vascular endothelial cells on a substrate to form a vascular endothelial cell layer, seeding neural stem cells on the vascular endothelial cell layer to make the neural stem cells be physically in direct contact with the vascular endothelial cell layer, and culturing the neural stem cells and the vascular endothelial cell layer to differentiate into neurons and branching vasculatures to form a cell sheet construct.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/826,927, filed on Aug. 14, 2015, and claims priority under 35 U.S.C.§119(a) on Patent Application No. 103128059 filed in Taiwan, R.O.C. onAug. 15, 2014, the entire contents of which are hereby incorporated byreference.

Some references, if any, which may include patents, patent applicationsand various publications, may be cited and discussed in the descriptionof this invention. The citation and/or discussion of such references, ifany, is provided merely to clarify the description of the presentinvention and is not an admission that any such reference is “prior art”to the invention described herein. All references listed, cited and/ordiscussed in this specification are incorporated herein by reference intheir entireties and to the same extent as if each reference wasindividually incorporated by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The invention relates to a cell sheet construct for neurovascularreconstruction, particularly to a cell sheet construct that is formed byco-culturing vascular endothelial cells with neural stem cells beingphysically in direct contact with each other to differentiate intobranching vasculatures and neurons of the cell sheet construct.

2. Description of the Related Art

Cell-based therapies have been emerging as a promising therapeuticstrategy for treating damaged or diseased organs and tissues. However,when being applied to the highly complicated nervous system, such asstroked brains, cell-based therapies remain to be improved further. Inorder to restore function of the damaged central nervous system bycell-based therapies, neurons have to be located in an appropriatemicroenvironment in the brain to transmit neural signals. Since neurons,which transmit neural signals, are regulated by vascular endothelialcells and interact with each other, neurons have to be situated in theneurovascular unit (NVU) to function appropriately.

Some researchers tried to create regenerated neurovascular tissue invitro by the methods of tissue engineering. However, these teams wereunable to create cell sheet having neurovascular tissues by co-culturingneural stem cells and vascular endothelial cells [Hicks C, Stevanato L,Stroemer R P, Tang E, Richardson S, Sinden J. In vivo and in vitrocharacterization of the angiogenic effect of CTX0E03 human neural stemcells. Cell transplantation. 2013; 22:1541-1552]. The main difficulty ofthese researches is that the suitable conditions for differentiation ofneural stem cells and vascular endothelial cells are different. Forexample, Hicks et al. used Matrigel as the substrate for vascularendothelial cells to form branching vasculature tissues, but it wasunable to provide neural stem cells an appropriate microenvironment todifferentiate into neurons. In addition, because Matrigel is a mixture,Matrigel from different manufactures and different batches has differentingredients, which cannot provide a consistent condition to inducedifferentiation of neural stem cells.

Therefore, it is necessary to develop the techniques of constructing theneurovascular tissue, a cell sheet of neural stem cells-vascularendothelial cells (NSC-EC) having differentiated neurons and branchingvasculatures, in order to promote neurovascular reconstruction.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a cell sheet construct forneurovascular reconstruction, comprising a vascular endothelial celllayer and a neural stem cell layer. The vascular endothelial cell layerhas vascular endothelial cells, and the neural stem cell layer hasneural stem cells. The vascular endothelial cell layer is physically indirect contact with the neural stem cell layer, the vascular endothelialcell layer differentiates into branching vasculatures, and the neuralstem cell layer differentiates into neurons.

Another aspect of the present invention provides a method formanufacturing the above-mentioned cell sheet construct in vitro,comprising the steps of: culturing vascular endothelial cells on asubstrate to form a vascular endothelial cell layer; seeding neural stemcells on the vascular endothelial cell layer to ensure the neural stemcells being physically in direct contact with the vascular endothelialcell layer; and co-culturing the neural stem cells and the vascularendothelial cell layer to differentiate into neurons and branchingvasculatures to form a cell sheet construct.

Another aspect of the present invention provides a method forneurovascular reconstruction in vivo, comprising placing theabove-mentioned cell sheet construct on a target site whereneurovascular reconstruction is required for treating damaged ordiseased tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment, and wherein:

FIG. 1A to FIG. 1D show immunocytochemistry of cell samples in Example 3stained with mouse anti-GFAP antibody(green), rabbit anti-CD31 antibody(red), and DAPI (blue). The cell sample in FIG. 1A is monoculture ofhuman cerebral neural stem cells (hCNSC) (Control group 1), the cellsample in FIG. 1B is monoculture of human cerebral microvascularendothelial cell (hCMEC) (Control group 2), the cell sample in FIG. 1Cis co-culture of human cerebral neural stem cells (hCNSC) and humancerebral microvascular endothelial cells (hCMEC) with a Transwell®permeable support located between these two types of cells (Controlgroup 3), and the cell sample in FIG. 1D is the cell sheet construct ofneural stem cells-vascular endothelial cells (NSC-EC) obtained inExample 3.

FIG. 2A to FIG. 2F show immunocytochemistry of cell samples in Example4. FIG. 2A shows the immunocytochemistry of monoculture of humancerebral neural stem cells (hCNSC) (Control group 1) stained with mouseanti-MAP2 antibody (green) and DAPI (blue). FIG. 2B shows theimmunocytochemistry of monoculture of human cerebral microvascularendothelial cell (hCMEC) (Control group 2) stained with mouse anti-ZO1(green) and DAPI (blue). FIG. 2C shows the cell sheet construct ofneural stem cells-vascular endothelial cells (NSC-EC) without transferobtained in Example 3 (7-day culture group) stained with mouse anti-MAP2antibody (green), rabbit anti-CD31 antibody (red), and DAPI (blue). FIG.2D shows the cell sheet construct of neural stem cells-vascularendothelial cells (NSC-EC) without transfer obtained in Example 3 (7-dayculture group) stained with mouse anti-ZO1 (green), rabbit anti-CD31antibody (red), and DAPI (blue). FIG. 2E shows the cell sheet constructof neural stem cells-vascular endothelial cells (NSC-EC) with transferobtained in Example 4 (2+5-day culture group) stained with mouseanti-MAP2 antibody (green), rabbit anti-CD31 antibody (red), and DAPI(blue). FIG. 2F shows the cell sheet construct of neural stemcells-vascular endothelial cells (NSC-EC) with transfer obtained inExample 4 (2+5-day culture group) stained with mouse anti-ZO1 (green),rabbit anti-CD31 antibody (red), and DAPI (blue).

FIG. 3 shows statistical analyses of the immunocytochemistry in Example4.

FIG. 4A to FIG. 4F show immunocytochemistry of the cell sheet constructof neural stem cells-vascular endothelial cells (NSC-EC) obtained inExample 3. FIG. 4A shows the cell sheet construct stained with chickenanti-laminin antibody (green) and DAPI (blue). FIG. 4B shows the cellsheet construct stained with mouse anti-fibronectin antibody (green) andDAPI (blue). FIG. 4C shows the cell sheet construct stained with sheepanti-hyaluronic acid antibody (green) and DAPI (blue). FIG. 4D shows thecell sheet construct stained with mouse anti-vitronectin antibody(green) and DAPI (blue). FIG. 4E shows the cell sheet construct stainedwith rabbit anti-collagen I antibody (green) and DAPI (blue). FIG. 4Fshows the cell sheet construct stained with goat anti-collagen IVantibody (green) and DAPI (blue).

FIG. 5A to FIG. 5D show immunocytochemistry of the cortical lesiongrafted with the cell sheet constructs of neural stem cells-vascularendothelial cells (NSC-EC) of the present invention. FIG. 5A and FIG. 5Bshow the sample stained with mouse anti-MAP2 antibody (green), rabbitanti-CD31 antibody (red), and DAPI (blue). FIG. 5B is an enlarged viewwithin the white box in FIG. 5A. FIG. 5C and FIG. 5D show the samplestained with rabbit anti-HuN antibody (green), mouse anti-Nestinantibody (red), and DAPI (blue). FIG. 5D is an enlarged view within thewhite box in FIG. 5C.

FIG. 6A to FIG. 6C show the results of behavioral assessments and bodymeasurements of neurovascular reconstruction in rats with inducedtraumatic brain injury in Example 7. FIG. 6A shows the modifiedneurological severity score (mNSS) of the rats. FIG. 6B shows theresults of contralateral swing test. FIG. 6C shows the average bodyweight of the rats after brain injury and treatment.

FIG. 7 shows the results of Von Frey test for mice receiving spinalnerve ligation and grafting of the neural stem cell-vascular endothelialcell (NSC-EC) sheet in Example 8.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a cell sheet construct for neurovascularreconstruction. The cell sheet comprises a vascular endothelial celllayer and a neural stem cell layer. The vascular endothelial cell layerhas vascular endothelial cells, and the neural stem cell layer hasneural stem cells. The vascular endothelial cell layer is directly incontact with the neural stem cell layer, and the two layers directlyinteract with each other. The vascular endothelial cell layer providescellular source and extracellular matrix that are needed indifferentiation of the neural stem cell layer cells, so that the neuralstem cell layer differentiates into neurons, and in return, the vascularendothelial cell layer differentiates into branching vasculatures, whichprovide nutrient to the neural stem cells.

As used herein, the term “vascular endothelium” refers to the thin layerof simple squamous cells that lines the interior surface of bloodvessels. The thin layer of cells form an interface between circulatingblood and the rest of the vessel wall. The term “vascular endothelialcells” refers to the cells forming vascular endothelium, including, butnot limited to, coronary artery endothelial cells, aortic endothelialcells, cerebral microvascular endothelial cells, umbilical veinendothelial cells, and vascular endothelial cells in dermis. In someembodiments, the vascular endothelial cells are human cerebralmicrovascular endothelial cells. Vascular endothelial cells used in thepresent invention are commercially available, for example, but notlimited to human coronary arterial endothelial cells (CC-2585, Lonza),human brain microvascular endothelial cells (00194607, Lonza), humanaortic endothelial cells (CC-2535, Lonza), human brain microvascularendothelial cells (ACBRI 376, Cell Systems), human umbilical veinendothelial cells (C2519A), and human dermal microvascular endothelialcells (CC-2543).

As used herein, the term “neural stem cells” refers to self-renewing,multipotent cells that generate the main phenotype of the nervoussystem. Neural stem cells primarily differentiate into neurons,astrocytes, and oligodendrocytes. Neural stem cells include, but are notlimited to cerebral neural stem cells, neural crest stem cells, centralnervous system stem cells (CNS-SCs), and peripheral neural stem cells.In some embodiments, the neural stem cells are human cerebral neuralstem cells. Neural stem cells used in the present invention arecommercially available, for example, but not limited to human cortexneural progenitor cell line (SCC007, Millipore), human neural crest stemcell microbeads (130-097-127, Miltenyl Biotec), and human ventralmidbrain neural progenitor cell line (SCC008, Millipore).

In some embodiments, the vascular endothelial cell layer furthercomprises extracellular matrix. The extracellular matrix and thevascular endothelial cells can be used as a carrier for neural stem cellgrafting. The extracellular matrix is synthesized and secreted by thevascular endothelial cells and is a collection of marcomoleculesdistributing between cell surfaces and/or cells. These marcomoleculesform a complicate network structure, support and connect tissues, andregulate development of tissues and cellular activities. Theextracellular matrix includes polypeptide, collagen, polysaccharide,hyaluronic acid, fibronectin, vitronectin, laminin, and their mixture.

The cell sheet construct of the present invention can further betransferred to a target location and continue differentiating intoneurons and branching vasculatures. The target location includes, but isnot limited to, neurovascular tissues of limbs, cerebrum, cerebellum,medulla oblongata, spinal cord, heart, lung, liver, stomach, smallintestine, large intestine, colon, kidney, gallbladder, pancreas, anduterus. In some embodiments, the target location is cerebral cortex orspinal nerves of a mammal. In some embodiments, the mammal is a rat,mouse, goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, orhuman.

The present invention also provides a novel method for manufacturing theabove-mentioned cell sheet construct for neurovascular reconstruction.The formation of the cell sheet construct depends on direct contact andinteraction between neural stem cells and vascular endothelial cells. Amonolayer of vascular endothelial cells is used as biomaterial insupport of cultivating neural stem cells and increase neuronaldifferentiation of the neural stem cells, and the neural stem cells alsofacilitate branching morphogenesis of the vascular endothelial cells, sothat a cell sheet of neural stem cells-vascular endothelial cells(NSC-EC) functioning as neurovascular tissue is formed. The methodcomprises the steps of: culturing vascular endothelial cells on asubstrate to form a vascular endothelial cell layer; seeding neural stemcells on the vascular endothelial cell layer to make the neural stemcells be physically in direct contact with the vascular endothelial celllayer; and co-culturing the neural stem cells and the vascularendothelial cell layer to differentiate into neurons and branchingvasculatures to form a cell sheet construct.

In some embodiments, the substrate contains collagen, which comprises,but is not limited to type Ito type XXVIII collagen. In someembodiments, the collagen is type I collagen.

In some embodiments, the cell density of the vascular endothelial celllayer is about 100,000 cells/cm² to 300,000 cells/cm². In someembodiments, the neural stem cells are seeded with a density of about10,000 cells/cm² to 100,000 cells/cm².

In some embodiments, the vascular endothelial cells are cultured in avascular endothelial cell medium. In some embodiments, the vascularendothelial cell medium is an endothelial cell growth-2 (EGM™-2) mediumcomprising 1-10% (v/v) fetal bovine serum (FBS), 0.1-5% (v/v) 10,000U/mL penicillin, and 0.1-5% (v/v) 10,000 U/mL streptomycin.

In some embodiments, the neural stem cells are physically in directcontact with the vascular endothelial cell layer and co-cultured in aneural stem cell/endothelial cell co-culture medium. In someembodiments, the neural stem cell/endothelial cell co-culture medium isserum-free, and by co-culturing the neural stem cells and the vascularendothelial cells, the two types of cells facilitate differentiation ofthe other type of cells. In some embodiments, the neural stemcell/endothelial cell co-culture medium contains 50% (v/v) DMEM/F-12medium and 50% (v/v) EGM™-2 medium and 0.001-0.03% (v/v) human albuminsolution, 100-5000 μg/ml transferrin, 0.1-100 μg/ml putrescine DiHC1,0.1-10 μg/ml insulin, 1-100 ng/ml progesterone 0.1-10 mM L-glutamine,and 1-100 ng/ml sodium selenite.

In some embodiments, the method further comprises transferring the cellsheet construct to a new substrate and keeping culturing the cell sheetconstruct to increase neuronal differentiation of the neural stem cellsand maintain formation of branching vasculatures and differentiation ofendothelial cells. In some embodiments, the vascular endothelial cellsand the neural stem cells are cultured on a temperature-responsive cellculture surface to form a cell sheet of neural stem cells-vascularendothelial cells (NSC-EC). Then, the cell sheet is detached from thetemperature-responsive cell culture surface at room temperature (about20 to 25° C.), and the detached cell sheet is transferred onto a newsubstrate through a transfer membrane to keep growing. Neuronaldifferentiation of the neural stem cells is increased more than 2-fold,and the functions of neurovascular tissue of the cell sheet areimproved.

In some embodiments, the new substrate contains collagen, whichcomprises, but is not limited to type Ito type XXVIII collagen. In someembodiments, the collagen is type I collagen.

The present invention also provides a novel method for neurovascularreconstruction in vivo, comprising placing the above-mentioned cellsheet construct on a target site where neurovascular reconstruction isrequired for treating damaged or diseased tissue.

In some embodiments, the cell sheet construct is transferred from aculture surface to the target site with a transfer membrane.

In some embodiments, the target sites include, but is not limited to,limbs, cerebrum, cerebellum, medulla oblongata, spinal cord, heart,lung, liver, stomach, small intestine, large intestine, colon, kidney,gallbladder, pancreas, and uterus. In some embodiments, the target siteis cerebrum or spinal nerves.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a component” includes a plurality of suchcomponents and equivalents thereof known to those skilled in the art.

As used herein, “around”, “about” or “approximately” shall generallymean within 20 percent, preferably within 10 percent, and morepreferably within 5 percent of a given value or range. Numericalquantities given herein are approximate, meaning that the term “around”,“about” or “approximately” can be inferred if not expressly stated.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person skilled in theart to which this invention belongs.

The present invention is described in more detail in the followingillustrative examples. Although the examples may represent only selectedembodiments of the invention, it should be understood that the followingexamples are illustrative and not limiting.

EXAMPLE 1 Culture of Human Cerebral Neural Stem Cell (hCNSC) Line

The following materials for cell culture are commercially available.Human cerebral neural stem cells (hCNSC) were purchased from Millipore(human cortex neural progenitor cell line, SCC007, Millipore, Mass.,USA). Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12,transferrin, putrescine DiHC1, insulin, progesterone, L-glutamine,sodium selenite, 4-hydroxytamoxifen, and Accutase were all purchasedfrom Sigma-Aldrich Corporation (MO, USA). Human albumin solution waspurchased from GemBio Laboratory (CA, USA), and human recombinantepidermal growth factor (EGF) and basic fibroblast growth factor (bFGF)were both purchased from PeproTech Inc. (NJ, USA).

Human cerebral neural stem cells (hCNSC) were seeded on culture dishesand maintained in DMEM/F12 medium containing 0.03% human albuminsolution, 100 μg/ml transferrin, 16.2 μg/ml putrescine, 5μg/ml insulin,60 ng/ml progesterone, 2 mM L-glutamine, 40 ng/ml sodium selenite, 10ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growthfactor (EGF), and 100 nM 4-hydroxytamoxifen, at 37° C., 5% CO₂ for 48hours. Then, human cerebral neural stem cells (hCNSC) were detached fromthe culture dishes by treatment of Accutase, and the hCNSC cellsuspension was collected for co-cultur with human cerebral microvascularendothelial cells (hCMEC).

EXAMPLE 2 Culture of Human Cerebral Microvascular Endothelial Cell(hCMEC) Line

The following materials for cell culture are commercially available.Human cerebral microvascular endothelial cells (hCMEC) and EGM™-2culture medium were purchased from Lonza Biologics (00194607, Lonza,N.J., USA). Fetal bovine serum (FBS) was purchased from PAA Laboratories(ON, Canada). Antibiotics (containing 10,000U/mL penicillin and 10,000U/mL streptomycin) were purchased from Invitrogen (CA, USA).

Human cerebral microvascular endothelial cells (hCMEC) were seeded onculture dishes and maintained in EGM™-2 medium containing 5% FBS and 1%antibiotics solution mentioned above, at 37° C., 5% CO₂ for 48 hours.After that, human cerebral microvascular endothelial cells (hCMEC) weredetached from the culture dishes by treatment of Accutase, and the hCMECsuspension was collected for co-cultur with human cerebral neural stemcells (hCNSC).

EXAMPLE 3

Co-Culture of Human Cerebral Neural Stem Cell (hCNSC) Line and HumanCerebral Microvascular Endothelial Cell (hCMEC) Line

Human cerebral microvascular endothelial cell (hCMEC) suspensionobtained in Example 2 was seeded on cell culture dishes (BD Biosciences,USA) coated with collagen type I and maintained in EGM™-2 mediumcontaining 5% FBS and 1% antibiotics solution (containing 10,000U/mLpenicillin and 10,000U/mL streptomycin), at 37° C., 5% CO₂ until thecells reached about 200,000 cells/cm² and formed a vascular endothelialcell layer. After that, human cerebral neural stem cell (hCNSC)suspension obtained in Example 1 was seeded on the vascular endothelialcell layer at a density of about 5,000 cells/cm², so that the humancerebral neural stem cell (hCNSC) and the human cerebral microvascularendothelial cell (hCMEC) are physically in direct contact and interactwith each other. The two types of cells were co-cultured in the neuralstem cell/endothelial cell co-culture medium (containing 50% (v/v)DMEM/F-12 medium and 50% (v/v) EGM™-2 medium and 0.015% (v/v) humanalbumin solution, 500 μg/ml transferrin, 8.1 μg/ml putrescine DiHC1, 2.5μg/ml insulin, 30 ng/ml progesterone, 1 mM L-glutamine, and 20 ng/mlsodium selenite) at 37° C., 5% CO₂ for 7 days. Cell culture medium wasreplaced every other day until branching vasculatures, neurons and otherneural lineage cells were differentiated to form a cell sheet constructof neural stem cells-vascular endothelial cells (NSC-EC). The cell sheetconstruct was fixed with 4% paraformaldehyde (PFA) for 10 minutes,rinsed with phosphate buffered saline (PBS), and stored in PBS at 4° C.for immunocytochemistry analysis.

In addition, monoculture of human cerebral neural stem cells (hCNSC)(Control group 1), monoculture of human cerebral microvascularendothelial cell (hCMEC) (Control group 2), and co-culture of humancerebral neural stem cells (hCNSC)and human cerebral microvascularendothelial cell (hCMEC) with a Transwell® permeable support (CorningInc., USA) located between these two types of cells (Control group 3)were used to compare cell differentiation in different treatments.

Monoculture of human cerebral neural stem cells (hCNSC) (Controlgroup 1) was carried out as follows. Human cerebral neural stem cell(hCNSC) suspension obtained in Example 1 was seeded on culture dishescoated with laminin (Sigma-Aldrich Corporation, USA) at a density ofabout 25,000 cells/cm² in the neural stem cell/endothelial cellco-culture medium (containing 50% (v/v) DMEM/F-12 medium and 50% (v/v)EGM™-2 medium and 0.015% (v/v) human albumin solution, 500 μg/mltransferrin, 8.1 μg/ml putrescine DiHC1, 2.5 μg/ml insulin, 30 ng/mlprogesterone, 1 mM L-glutamine, and 20 ng/ml sodium selenite) at 37° C.,5% CO₂. Cell culture medium was replaced every other day. After beingcultured for 7 days, the cells were fixed with 4% paraformaldehyde (PFA)for 10 minutes, rinsed with PBS, and stored in PBS at 4° C. forimmunocytochemistry analysis.

Monoculture of human cerebral microvascular endothelial cells (hCMEC)(Control group 2) was carried out as follows. Human cerebralmicrovascular endothelial cell (hCMEC) suspension obtained in Example 2was seeded on culture dishes coated with collagen type I (BDBiosciences, USA) at a density of about 40,000 cells/cm² in the neuralstem cell/endothelial cell co-culture medium (containing 50% (v/v)DMEM/F-12 medium and 50% (v/v) EGM™-2 medium and 0.015% (v/v) humanalbumin solution, 500 μg/ml transferrin, 8.1 μg/ml putrescine DiHCl, 2.5μg/ml insulin, 30 ng/ml progesterone, 1 mM L-glutamine, and 20 ng/mlsodium selenite) at 37° C., 5% CO₂. Cell culture medium was replacedevery other day. After being cultured for 7 days, the cells were fixedwith 4% paraformaldehyde (PFA) for 10 minutes, rinsed with PBS, andstored in PBS at 4° C. for immunocytochemistry analysis.

Co-culture of human cerebral neural stem cells (hCNSC) and humancerebral microvascular endothelial cells (hCMEC) with a Transwell®permeable support (Corning Inc., USA) located between these two types ofcells (Control group 3) was carried out as follows. Human cerebralneural stem cell (hCNSC) suspension obtained in Example 1 was seededinto Transwell® permeable supports (with 0.4 μm pore polyester membraneinsert, Corning Inc., USA) coated with laminin (Sigma-AldrichCorporation, USA) at a density of about 15,000 cells/cm² in DMEM/F12medium containing 0.03% human albumin solution, 100 μg/ml transferrin,16.2 μg/ml putrescine, 5 μg/ml insulin, 60 ng/ml progesterone, 2 mML-glutamine, 40 ng/ml sodium selenite, 10 ng/ml basic fibroblast growthfactor (bFGF), 20 ng/ml epidermal growth factor (EGF), and 100 nM4-hydroxytamoxifen, at 37° C., 5% CO₂, for proliferation to 80%confluency. Meanwhile, human cerebral microvascular endothelial cell(hCMEC) suspension obtained in Example 2 was seeded on culture dishescoated with collagen type I (BD Biosciences, USA) at a density of about40,000 cells/cm² in EGM-2 medium containing 5% FBS and 1% antibioticssolution (containing 10,000U/mL penicillin and 10,000U/mL streptomycin),at 37° C., 5% CO₂, for 7 days. The Transwell® permeable supportscontaining hCNSCs were then transferred to the top of the culture dishescontaining hCMECs, and the distance between the transwell membrane andthe bottom of the monolayer of hCMECs was about 850 μm. The cells werecultured together in the neural stem cell/endothelial cell co-culturemedium (containing 50% (v/v) DMEM/F-12 medium and 50% (v/v) EGM™-2medium and 0.015% (v/v) human albumin solution, 500 μ/ml transferrin,8.1 μg/ml putrescine DiHC1, 2.5 μg/ml insulin, 30 ng/ml progesterone, 1mM L-glutamine, and 20 ng/ml sodium selenite) at 37° C., 5% CO₂. Cellculture medium was replaced every other day. After being cultured for 7days, the cells were fixed with 4% paraformaldehyde (PFA) for 10minutes, rinsed with PBS, and stored in PBS at 4° C. forimmunocytochemistry analysis.

The cell sheet construct of neural stem cells-vascular endothelial cells(NSC-EC) obtained in this Example and cell samples of Control groups 1-3were then analyzed by immunocytochemistry. For immunocytochemistry,cells were blocked with 10% normal goat serum in PBS containing 0.1%Triton X-100 (Sigma) for 30 min prior to incubation with mouseanti-glial fibrillary acid protein (GFAP), which specifically detectsneural stem cells and astrocyte (1:3000; Sigma, USA), and rabbitanti-CD31/platelet endothelial cell adhesion molecule 1, whichspecifically detects endothelial cells representing the “vascularcomponent”(1:200; Abcam, UK), primary antibodies for 18 hat 4° C. Afterremoval of primary antibodies and washing with PBS (3x), cells wereincubated with appropriate secondary antibodies (goat anti-mouse Alexa488-labeled, 1:1000; goat anti-rabbit Alexa 555-labeled, 1:1000,Invitrogen, USA) for 1 h at room temperature (22° C.). Stainedcoverslips were rinsed in PBS and mounted with Vectashield forfluorescence with diamidino-2-phenylindole (DAPI, Vector Laboratories,USA) to detect cell nuclei. Fluorescence images were captured using afluorescence microscope (AxioImager M2, Zeiss) to analyzecytoarchitecture, including branching vasculatures and the proportion ofneurons and other neural lineage cells differentiated from neural stemcells. The results are shown in FIG. 1A to FIG. 1D.

FIG. 1A shows the immunocytochemistry of monoculture of human cerebralneural stem cells (hCNSC) (Control group 1) stained with mouse anti-GFAPantibody (specifically detects neural stem cells and astrocyte, green),rabbit anti-CD31 antibody (specifically detects endothelial cells, red),and DAPI (detects cell nuclei, blue). As shown in FIG. 1A, there are novascular endothelial cells in the monoculture of hCNSCs (Control group1).

FIG. 1B shows the immunocytochemistry of monoculture of human cerebralmicrovascular endothelial cell (hCMEC) (Control group 2) stained withmouse anti-GFAP antibody (specifically detects neural stem cells andastrocyte, green), rabbit anti-CD31 antibody (specifically detectsendothelial cells, red), and DAPI (detects cell nuclei, blue). As shownin FIG. 1B, there are no neural stem cells or branching vasculatures inthe monoculture of hCMECs (Control group 2).

FIG. 1C shows the immunocytochemistry of co-culture of human cerebralneural stem cells (hCNSC) and human cerebral microvascular endothelialcells (hCMEC) with a Transwell® permeable support (Corning Inc., USA)located between these two types of cells (Control group 3) stained withmouse anti-GFAP antibody (specifically detects neural stem cells andastrocyte, green), rabbit anti-CD31 antibody (specifically detectsendothelial cells, red), and DAPI (detects cell nuclei, blue). As shownin FIG. 1C, because hCNSCs and hCMECs are not physically in directcontact with each other, there are no differentiated branchingvasculatures, neural stem cells, or neural lineage cells in theco-culture (Control group 3).

FIG. 1D shows the immunocytochemistry of the cell sheet construct ofneural stem cells-vascular endothelial cells (NSC-EC) obtained in thisExample stained with mouse anti-GFAP antibody (specifically detectsneural stem cells and astrocyte, green), rabbit anti-CD31 antibody(specifically detects endothelial cells, red), and DAPI (detects cellnuclei, blue). As shown in FIG. 1D, the cell sheet construct of neuralstem cells-vascular endothelial cells (NSC-EC), which is formed bydirect contact and interaction of neural stem cells and vascularendothelial cells, has neural stem cells (stained in green) andbranching vasculatures (stained in red, having branched structures).

As the results shown in FIG. 1A to FIG. 1D, neural stem cells andvascular endothelial cells have to be physically in direct contact witheach other to form a cell sheet construct of neural stem cells-vascularendothelial cells (NSC-EC) having differentiated neural lineage cellsand branching vasculatures.

EXAMPLE 4 Transfer of Cell Sheet Constructs of Neural StemCells-Vascular Endothelial Cells (NSC-EC)

The culture dishes with temperature-responsive cell culture surfaces(Nunc® UpCell Surface) used in this Example were purchased from ThermoScientific (USA). The temperature-responsive cell culture surface isslightly hydrophobic at 37° C., allowing cells to attach and grow. Whenthe temperature of the culture is reduced to below 32° C., the surfacebecomes very hydrophilic, binds water, swells and releases adherentcells.

Human cerebral microvascular endothelial cell (hCMEC) suspensionobtained in Example 2 was seeded on culture dishes withtemperature-responsive cell culture surfaces (Nunc® UpCell Surface,Thermo Scientific, USA) in EGM™-2 medium containing 5% FBS and 1%antibiotics solution (containing 10,000 U/mL penicillin and 10,000U/mLstreptomycin), at 37° C., 5% CO₂ until the cells reached about 200,000cells/cm² and formed a vascular endothelial cell layer. After that,human cerebral neural stem cell (hCNSC) suspension obtained in Example 1was seeded on the vascular endothelial cell layer at a density of about5,000 cells/cm², so that the hCNSCs and the hCMECs are physically indirect contact and interact with each other. The two types of cells wereco-cultured in the neural stem cell/endothelial cell co-culture medium(containing 50% (v/v) DMEM/F-12 medium and 50% (v/v) EGM™-2 medium and0.015% (v/v) human albumin solution, 500 μg/ml transferrin, 8.1 μg/mlputrescine DiHC1, 2.5 μg/ml insulin, 30 ng/ml progesterone, 1 mML-glutamine, and 20 ng/ml sodium selenite) at 37° C., 5% CO₂ for 2 daysto form cell sheet constructs of neural stem cells-vascular endothelialcells (NSC-EC). After that, the culture dishes were placed at a 22° C.incubator to make the temperature-responsive cell culture surfacehydrophilic, and transfer membranes were covered on the NSC-EC cellsheet construct. About 20 minutes later, the NSC-EC cell sheetconstructs detached from the culture dishes and attached to the transfermembranes. The NSC-EC cell sheet constructs were then transferred toglass coverslips which had been coated with collagen at 37° C. for about30 minutes to allow the NSC-EC cell sheet constructs to attach to theglass coverslips. After the transfer membranes were removed, the NSC-ECcell sheet constructs were cultured in the neural stem cell/endothelialcell co-culture medium (containing 50% (v/v) DMEM/F-12 medium and 50%(v/v) EGM™-2 medium and 0.015% (v/v) human albumin solution, 500 μg/mltransferrin, 8.1 μg/ml putrescine DiHC1, 2.5 μg/ml insulin, 30 ng/mlprogesterone, 1 mM L-glutamine, and 20 ng/ml sodium selenite) at 37° C.,5% CO₂ for 5 more days. Cell culture medium was replaced every otherday. After being cultured for a total of 7 days, the cells were fixedwith 4% paraformaldehyde (PFA) for 10 minutes, rinsed with PBS, andstored in PBS at 4° C. for immunocytochemistry analysis.

In addition, monoculture of human cerebral neural stem cells (hCNSC)(Control group 1), monoculture of human cerebral microvascularendothelial cell (hCMEC) (Control group 2), and the cell sheetconstructs of neural stem cells-vascular endothelial cells (NSC-EC)without transfer obtained in Example 3 (7-day culture group) were usedto compare cell differentiation in different treatments with the cellsheet constructs of neural stem cells-vascular endothelial cells(NSC-EC) with transfer obtained in this Example (Example 4) (2+5-dayculture group). Cell culture of Control group 1, Control group 2, and7-day culture group was described in Example 3.

Immunocytochemistry was described in Example 3, with antibodies of mouseanti-microtubule associate protein-2 (MAP2), which specifically detectsneurons (1:500; Abcam, UK), mouse anti-zonula occludens 1 (Z01), whichspecifically detects vascular endothelial cells (1:500; Zymed, USA), andrabbit anti-CD31/platelet endothelial cell adhesion molecule 1, whichspecifically detects endothelial cells representing the “vascularcomponent” (1:200; Abcam, UK). Cell samples were mounted withVectashield for fluorescence with diamidino-2-phenylindole (DAPI, VectorLaboratories, USA) to detect cell nuclei.

All experiments consisted of 3 biological replicates, each consisting of3 technical replicates. For each technical replicate, 5 images weretaken from different areas on the coverslip prior to calculating a meanvalue of cell counts for each coverslip. Using SPSS 17 for Mac (IBM,USA), a non-parametric Kruskall-Wallis was used to compare differentconditions followed by a Dunn's post- hoc comparison to determine whichconditions were significantly different (p<0.05). A Mann-Whitney U testwas used to compare the markers of differentiation between mono- andco-cultures. Graphs were drawn in Prism 5 (GraphPad, USA) with datapoints representing the median and bars reflecting the value range.

The results are shown in FIG. 2A to FIG. 2F. FIG. 2A shows theimmunocytochemistry of monoculture of human cerebral neural stem cells(hCNSC) (Control group 1) stained with mouse anti-MAP2 antibody(specifically detects neurons, green) and DAPI (detects cell nuclei,blue). As shown in FIG. 2A, there are only few neurons in themonoculture of human cerebral neural stem cells (hCNSC) (Control group1).

FIG. 2B shows the immunocytochemistry of monoculture of human cerebralmicrovascular endothelial cell (hCMEC) (Control group 2) stained withmouse anti-ZO1 (specifically detects vascular endothelial cells, green)and DAPI (detects cell nuclei, blue). As shown in FIG. 2B, there are nobranching vasculatures in the monoculture of hCMECs (Control group 2).

FIG. 2C shows the cell sheet constructs of neural stem cells-vascularendothelial cells (NSC-EC) without transfer obtained in Example 3 (7-dayculture group) stained with mouse anti-MAP2 antibody (specificallydetects neurons, green), rabbit anti-CD31 antibody (specifically detectsendothelial cells, red), and DAPI (detects cell nuclei, blue). As shownin FIG. 2C, the NSC-EC cell sheet constructs without transfer obtainedin Example 3 (7-day culture group) has differentiated neurons (green)and branching vasculatures (red). FIG. 2D shows the NSC-EC cell sheetconstructs without transfer obtained in Example 3 (7-day culture group)stained with mouse anti-ZO1 (specifically detects vascular endothelialcells, green), rabbit anti-CD31 antibody (specifically detectsendothelial cells, red), and DAPI (detects cell nuclei, blue). As shownin FIG. 2D, the NSC-EC cell sheet constructs without transfer obtainedin Example 3 (7-day culture group) has differentiated branchingvasculatures (green and red).

FIG. 2E shows the cell sheet constructs of neural stem cells-vascularendothelial cells (NSC-EC) with transfer obtained in Example 4 (2+5-dayculture group) stained with mouse anti-MAP2 antibody (specificallydetects neurons, green), rabbit anti-CD31 antibody (specifically detectsendothelial cells, red), and DAPI (detects cell nuclei, blue). As shownin FIG. 2E, the NSC-EC cell sheet constructs with transfer obtained inExample 4 (2+5-day culture group) has differentiated neurons (green) andbranching vasculatures (red), and compared to the 7-day culture group,the amount of differentiated neurons (green) in the 2+5-day culturegroup significantly increased. FIG. 2F shows the NSC-EC cell sheetconstructs with transfer obtained in Example 4 (2+5-day culture group)stained with mouse anti-ZO1 (specifically detects vascular endothelialcells, green), rabbit anti-CD31 antibody (specifically detectsendothelial cells, red), and DAPI (detects cell nuclei, blue). As shownin FIG. 2F, the NSC-EC cell sheet constructs with transfer obtained inExample 4 (2+5-day culture group) has differentiated branchingvasculatures (green and red).

FIG. 3 shows statistical analyses of the immunocytochemistry. Neuronaldifferentiation (MAP2+ cells) of human cerebral neural stem cell (hCNSC)significantly increased from 8% (monoculture of hCNSCs, Control group 1)to 31% (cell sheet constructs of neural stem cells-vascular endothelialcells (NSC-EC) without transfer obtained in Example 3, 7-day culturegroup) and 64% (NSC-EC cell sheet constructs with transfer obtained inExample 4, 2+5-day culture group), respectively, whereas differentiationof vascular endothelial cells (ZO1+ cells) did not increasesignificantly. The results show that the NSC-EC cell sheet constructs ofthe present invention increases neuronal differentiation of neural stemcells.

EXAMPLE 5 Analysis of Extracellular Matrix of the Cell Sheet Constructsof Neural Stem Cells-Vascular Endothelial Cells (NSC-EC) of the PresentInvention

Extracellular matrix of the cell sheet constructs of neural stemcells-vascular endothelial cells (NSC-EC) obtained in Example 3 wasanalyzed by immunocytochemistry. Immunocytochemistry was described inExample 3, with antibodies of chicken anti-laminin (1:500, Abcam, USA),mouse anti-fibronectin (1:200, Abcam, USA), sheep anti-hyaluronic acid(1:100, Abcam, USA), mouse anti-vitronectin (1:1000, Abcam, USA), rabbitanti-collagen I (1:500, Abcam, USA), and goat anti-collagen IV (1:200,Millipore, USA). Cell samples were also mounted with Vectashield forfluorescence with diamidino-2-phenylindole (DAPI, Vector Laboratories,USA) to detect cell nuclei. The results are shown in FIG. 4A to 4F.

FIG. 4A shows the cell sheet constructs stained with chickenanti-laminin antibody (specifically detects laminin, green) and DAPI(detects cell nuclei, blue). As shown in FIG. 4A, the cell sheetconstructs of neural stem cells-vascular endothelial cells (NSC-EC) ofthe present invention has a great amount of laminin (green).

FIG. 4B shows the cell sheet constructs stained with mouseanti-fibronectin antibody (specifically detects fibronectin, green) andDAPI (detects cell nuclei, blue). As shown in FIG. 4B, the cell sheetconstructs of neural stem cells-vascular endothelial cells (NSC-EC) ofthe present invention contains fibronectin (green).

FIG. 4C shows the cell sheet constructs stained with sheepanti-hyaluronic acid antibody (specifically detects hyaluronic acid,green) and DAPI (detects cell nuclei, blue). As shown in FIG. 4C, thecell sheet constructs of neural stem cells-vascular endothelial cells(NSC-EC) of the present invention has a great amount of hyaluronic acid(green).

FIG. 4D shows the cell sheet constructs stained with mouseanti-vitronectin antibody (specifically detects vitronectin, green) andDAPI (detects cell nuclei, blue). As shown in FIG. 4D, the cell sheetconstructs of neural stem cells-vascular endothelial cells (NSC-EC) ofthe present invention has a great amount of vitronectin (green).

FIG. 4E shows the cell sheet constructs stained with rabbitanti-collagen I antibody (specifically detects collagen I, green) andDAPI (detects cell nuclei, blue). As shown in FIG. 4E, the cell sheetconstructs of neural stem cells-vascular endothelial cells (NSC-EC) ofthe present invention has a great amount of collagen I (green).

FIG. 4F shows the cell sheet constructs stained with goat anti-collagenIV antibody (specifically detects collagen IV, green) and DAPI (detectscell nuclei, blue). As shown in FIG. 4F, the cell sheet constructs ofneural stem cells-vascular endothelial cells (NSC-EC) of the presentinvention contains collagen IV (green).

The results shown in FIG. 4A to FIG. 4F indicate that extracellularmatrix of the cell sheet constructs of neural stem cells-vascularendothelial cells (NSC-EC) of the present invention contains at leastcollagen, hyaluronic acid, fibronectin, vitronectin, and laminin.

EXAMPLE 6 Application of Cell Sheet Constructs of Neural StemCells-Vascular Endothelial Cells (NSC-EC) on NeurovascularReconstruction

Adult male Sprague-Dawley (SD) rats weighing 300˜350 g were anesthetizedwith ketamine (75 mg/kg, intraperitoneal injection, i.p.) and xylazine(10 mg/kg, i.p.), supplemented with ketamine (20 mg/kg, i.p.) as needed.Anesthetized rats were then secured in a stereotactic frame, and thescalp was incised along the midline. The right side of the skull wasremoved (size of craniotomy, about 1 cm²) using a drill and a rongeur.The coordinates of the three points from the bregma were 4 mm rostral/1mm lateral (coordinate A=+4, +1), 2 mm caudal/1 mm lateral (coordinateB=−2, +1), and 4 mm rostral/6 mm lateral (coordinate C=+4,+6). For thecorticectomy, the underlying cortex was sharply cut out with a surgicalblade to a depth of 4 mm. Then, the cell sheet constructs of neural stemcells-vascular endothelial cells (NSC-EC) of the present invention(about 1 cm²) was placed onto the cortical lesion with transfermembranes. About 30 minutes later, after the cell sheet constructsdetached from the transfer membranes and attached to the surface of thecortical lesion, the transfer membranes were removed, and pia mater,arachnoid mater, and dura mater were sutured. The removed skulls wereplaced back with bone cement. Seven (7) days later, the rats' brainswere fixed with 4% of paraformaldehyde by transcardial perfusion.Cryostat section samples of the fixed brains were then analyzed byimmunocytochemistry with mouse anti-MAP2 antibody (specifically detectsneurons), rabbit anti-CD31 antibody (specifically detects endothelialcells), mouse anti-Nestin antibody (specifically detects neural stemcells), rabbit anti-HuN antibody (specifically detects human cellnuclei), and DAPI (detects cell nuclei). The results were shown in FIG.5A to FIG. 5D.

FIG. 5A and FIG. 5B show the cortical lesion covered with the cell sheetconstructs of neural stem cells-vascular endothelial cells (NSC-EC) ofthe present invention stained with mouse anti-MAP2 antibody(specifically detects neurons, green), rabbit anti-CD31 antibody(specifically detects endothelial cells, red), and DAPI (detects cellnuclei, blue). There are neurons differentiated from neural stem cells(green parts in FIG. 5A and FIG. 5B) and vascular endothelial cells (redparts in FIG. 5A and FIG. 5B) at the location where the cell sheetconstructs were implanted.

In addition, FIG. 5C and FIG. 5D show the cortical lesion covered withthe cell sheet constructs of neural stem cells-vascular endothelialcells (NSC-EC) of the present invention stained with rabbit anti-HuNantibody (specifically detects human cell nuclei, green), mouseanti-Nestin antibody (specifically detects neural stem cells, red), andDAPI (detects cell nuclei, blue). As shown in FIG. 5C and FIG. 5D, humancells (green parts) grew in the cortical lesion of rat brains,indicating that the neurons differentiated from neural stem cells aswell as the vascular endothelial cells in FIG. 5A and FIG. 5B came fromthe NSC-EC cell sheet constructs of the present invention.

As indicated in FIG. 5A to FIG. 5D, the cell sheet constructs of neuralstem cells-vascular endothelial cells (NSC-EC) of the present inventioncan be used in transplantion surgery and reconstruct neurovasculartissues at lesion sites.

EXAMPLE 7 Evaluation of Neurovascular Reconstruction in InducedTraumatic Brain Injury with Cell Sheet Constructs of Neural StemCells-Vascular Endothelial Cells (NSC-EC)

Adult male Sprague-Dawley (SD) rats weighing 300˜350 g were anesthetizedwith ketamine (75 mg/kg, intraperitoneal injection, i.p.) and xylazine(10 mg/kg, i.p.), supplemented with ketamine (20 mg/kg, i.p.) as needed.Anesthetized rats were then secured in a stereotactic frame, and thescalp was incised along the midline. The right side of the skull wasremoved (size of craniotomy, about 1 cm²) using a drill and a rongeur.The coordinates of the three points from the bregma were 4 mm rostral/1mm lateral (coordinate A=+4, +1), 2 mm caudal/1 mm lateral (coordinateB=−2, +1), and 4 mm rostral/6 mm lateral (coordinate C=+4, +6). For thecorticectomy, the underlying cortex was sharply cut out with a surgicalblade to a depth of 4 mm. (1) In the brain injury group (Control group),after a craniotomy and exposure of a triangular window displaying theunderlying right frontoparietal area of the brain covered by the dura,the dura was carefully incised with a number 20 needle to minimizebleeding, before cutting of the underlying cortex as described above;(2) In the brain injury with NSC/EC cell sheet group (Treatment group),after the procedure for the control groups was carried out, a piece ofcell sheet of NSC/EC, with an area of about 1 cm², was placed onto thelesion cavity formed 20 minutes after tissue removal from thefrontoparietal area. For ensuring the attachment of the cell layer onthe brain tissue, duration of 1 to 30 minutes of placement of the sheetwas required before removing the UpCell™ transfer membrane (ThermoScientific, N.H., USA).

Modified Neurologic Severity Score (mNSS) was performed 1, 3, 7, 14, 21and 28 days after corticectomy by individuals blinded to theexperimental groups. mNSS consists of motor, sensory, balance, andreflex tests. Neurologic function is graded on a scale of 0-18 (normalscore 0; maximal deficit score 18, Table 1). One score point indicatesthe inability to perform the test or the lack of a reflex; thus, thehigher score, the more severe injury.

TABLE 1 Neurological severity score Modified neurological severityscores (mNSS)^(a) Points Motor test 6 Raising the rat by the tail 1Flexion of forelimb 1 Flexion of hind limb 1 Head moved more than 10° tothe vertical axis within 30 s Walking on the floor (normal = 0; maximum= 3) 0 Normal walk 1 Inability to walk straight 2 Circling toward theparetic side 3 Falling down to the paretic side Sensory tests 2 1Placing test (visual and tactile test) 2 Proprioceptive test (deepsensation, pushing the paw against the table edge to stimulate limbmuscles) Beam balance test (normal = 0; maximum = 6) 6 0 Balances withsteady posture 1 Grasps side of beam 2 Hugs the beam and one limb fallsdown from the beam 3 Hugs the beam and two limb falls down from thebeam, or spins on beam (>60s) 4 Attempts to balance on the beam butfalls off (>40 s) 5 Attempts to balance on the beam but falls off (>20s) 6 Falls off: no attempts to balance or hang on to the beam (<20 s)Reflexes absent and abnormal movements 4 1 Pinna reflex (head shake whenthe auditory meatus is touched) 1 Corneal reflex (eye blink when thecornea is lightly touched with cotton) 1 Startle reflex (motor responseto a brief noise from snapping a clipboard and paper) 1 Seizures,myocolonus, myodystony Maximum points 18 ^(a)One point is awarded forthe inability to perform a task or for the lack of a tested reflex. Ascore of 13-18 indicates severe injury; 7-12, moderate injury; 1-6, mildinjury.

The contralateral body swing test was used to assess bias in swingdirection in rats elevated by the base of the tail. Twenty separatetests were performed with the number of right and left initialhead/torso turns recorded. Prior to surgically induced brain injury,rats swing right and left with near equal frequency, leading to acontralateral ratio of 50%. Following corticectomy, rats with corticallesions have a biased swing to the side contralateral to the injury witha bias approaching 100%. The contralateral swing test was performed 1,3, 7, 14, 21 and 28 days after corticectomy by individuals blinded tothe experimental groups.

In addition, each mouse was weighed 1, 3, 7, 14, 21 and 28 days afterbrain injury.

The results are shown in FIG. 6A to FIG. 6C. FIG. 6A shows the mNSS ofthe rats. As shown in FIG. 6A, mNSS of the rats in the Treatment groupwas statistically lower than the Control group from Day 3. It indicatesthat neurological recovery in rats treated with cell sheettransplantation was greater than the rats in the control group.

FIG. 6B shows the results of contralateral swing test. As shown in FIG.6B, improvement in the contralateral swing test for rats in theTreatment group was statistically greater than the Control group fromDay 3. The rats treated with cell sheet transplantation had an improvedcontralateral ratio of 61%, in contrast with a ratio of 78% in theControl group.

FIG. 6C shows the average body weight of the rats after brain injury. Asshown in FIG. 6C, body weight of the rats in the Treatment groupincreased gradually and was higher than the weight before corticectomyon postoperative day 7 (Day 7). In contrast, rats in the Control groupdid not regain body weight until Day 14. Body weight of the rats in theTreatment group was statistically higher than Control group from Day 7.

EXAMPLE 8 Evaluation of Neurovascular Reconstruction in InducedTraumatic Spinal Injury with Cell Sheet Constructs of Neural StemCells-Vascular Endothelial Cells (NSC-EC)

Adult male C57BL/6J mice weighing 25-30 g were used in this study. Themice were anesthetized with sodium pentobarbital (80 mg/kg, i.p.), andthe hairs on their back were clipped. A midline incision above thelumbar spine exposed the left sixth lumbar transverse process. Thetransverse process was removed carefully with a small scraper. Theunderlying fifth lumbar (L5) nerve root was isolated and then tightlyligated with 8-0 nylon thread. Next, the wound was closed with 2-3muscle sutures (3-0 absorbable nylon suture) and 4-5 skin sutures (3-0non-absorbable nylon suture).

A 14-day schedule was used in this study. In the spinal nerve ligation(SNL) with NSC/EC cell sheet group (Treatment group), 20 minutes afterthe procedure for the control group was carried out, a piece of cellsheet of NSC/EC, with an area of about 1 cm², was placed surrounding thesegment of ligated spinal nerve (L5). For ensuring the attachment of thecell layer on the nerve, duration of 1 to 30 minutes of placement of thesheet was required before removing the UpCell™ transfer membrane (ThermoScientific, N.H., USA). Von Frey tests were performed prior and 3, 5, 7,10, and 13 days after SNL by individuals blinded to the experimentalgroups. After the last Von Frey test on day 13, the animals weresacrificed on day 14 for immunohistochemistry study. The number ofanimals in each group was at least 8 at the beginning of the experiment;and at least 5 mice in each group survived to complete this study.

Von Frey test for determination of mechanical allodynia is described asfollows. The mice were individually placed in a transparent acrylic box(9×9×15 cm) with a wire-mesh bottom and allowed to acclimate to theirenvironment for at least 30 min. The mechanical stimulus was appliedfrom underneath to the plantar aspect of the hind limb, with a gradualincrease in pressure by means of an Electronic von Frey apparatus (IITCInc., CA, USA). The end point was characterized by the removal of thepaw followed by clear flinching movements. After the paw withdrawal, theintensity of the pressure was automatically recorded. Each test wasrepeated 3 times with intervals of 5 minutes, and the average value wasused.

The data were expressed as means±S.E.M. Student's t test and one-wayANOVA were used to analyze the data. A difference was considered to besignificant atp<0.05.

The results of Von Frey test are shown in FIG. 7. The results of VonFrey test for the control group and treatment group indicate that theimprovement of paw withdrawal pressure in the group of NSC/EC sheettreatment was statistically greater than the control group from day 10after the surgery.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments are chosen and described in order to explain theprinciples of the invention and their practical application so as toactivate others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

What is claimed is:
 1. A method for neurovascular reconstruction,comprising placing a cell sheet construct on a target site whereneurovascular reconstruction is required for treating damaged ordiseased tissue, wherein the cell sheet construct is made by stepsconsisting of: step
 1. culturing vascular endothelial cells on a cellculture dish with a vascular endothelial cell medium to form a vascularendothelial cell layer; step
 2. seeding neural stem cells on thevascular endothelial cell layer to ensure the neural stem cells beingphysically in direct contact with the vascular endothelial cell layer;step
 3. co-culturing the neural stem cells and the endothelial celllayer to form a cell sheet construct of neural stem cells-vascularendothelial cells (NSC-EC); step
 4. detaching the cell sheet constructof NSC-EC from the culture dish and transferring the same to a cellculture surface; and step
 5. culturing the cell sheet construct ofNSC-EC with a NSC-EC co-culture medium.
 2. The method of claim 1,wherein the cell sheet construct is transferred from a culture surfaceto the target site with a transfer membrane.
 3. The method of claim 1,wherein the target site is selected from the group consisting of limbs,cerebrum, cerebellum, medulla oblongata, spinal cord, heart, lung,liver, stomach, small intestine, large intestine, colon, kidney,gallbladder, pancreas, and uterus.
 4. The method of claim 1, wherein theneural stem cells are human cerebral neural stem cell line, SCC007,Millipore.
 5. The method of claim 1, wherein the vascular endothelialcell layer is used as a carrier.
 6. The method of claim 1, wherein instep 1, the cell culture dish has a temperature-responsive cell culturesurface.
 7. The method of claim 1, wherein in step 1, the cell densityof the vascular endothelial cell layer is about 100,000 cells/cm² to300,000 cells/cm².
 8. The method of claim 1, wherein in step 2, theneural stem cells are seeded with a density of about 10,000 cells/cm² to100,000 cells/cm².
 9. The method of claim 1, wherein the vascularendothelial cell medium is an endothelial cell growth-2 (EGM™-2) mediumcomprising 1-10% (v/v) fetal bovine serum (FBS), 0.1-5% (v/v) 10,000U/mL penicillin and 0.1-5% (v/v) 10,000 U/mL streptomycin.
 10. Themethod of claim 1, wherein in step 3, the neural stem cells and theendothelial cell layer are co-cultured in a neural stem cell/endothelialcell co-culture medium.
 11. The method of claim 10, wherein the neuralstem cell/endothelial cell co-culture medium comprises 50% (v/v)DMEM/F-12 medium and 50% (v/v) EGM™-2 medium and 0.001-0.03% (v/v) humanalbumin solution, 100-5000 μg/ml transferrin, 0.1-100 μg/ml putrescineDiHC1, 0.1-10 μg/ml insulin, 1-100 ng/ml progesterone, 0.1-10 mML-glutamine, and 1-100 ng/ml sodium selenite.
 12. A method forneurovascular reconstruction, comprising placing a cell sheet constructon a target site where neurovascular reconstruction is required fortreating damaged or diseased tissue, wherein the cell sheet consists of:a vascular endothelial cell layer consisting of a layer of humancerebral microvascular endothelial cells and an extracellular matrix,wherein the layer of human cerebral microvascular endothelial cells isderived from isolated human adult brain tissue, and the extracellularmatrix is synthesized and secreted by the layer of cerebralmicrovascular endothelial cells, and the extracellular matrix consistsof polypeptides and polysaccharides; a neural stem cell layer consistingof a layer of cerebral neural stem cells derived from isolated fetalbrain tissue or embryonic stem cells, being physically in direct contactwith the vascular endothelial cell layer, and being on the vascularendothelial cell layer; branching vasculatures differentiated from thevascular endothelial cell layer; and neural lineage cells differentiatedfrom the neural stem cell layer.
 13. The method of claim 12, wherein thecell sheet construct is transferred from a culture surface to the targetsite with a transfer membrane.
 14. The method of claim 12, wherein thetarget site is selected from the group consisting of limbs, cerebrum,cerebellum, medulla oblongata, spinal cord, heart, lung, liver, stomach,small intestine, large intestine, colon, kidney, gallbladder, pancreas,and uterus.
 15. The method of claim 12, wherein the neural stem cellsare human cerebral neural stem cell line, SCC007, Millipore.
 16. Themethod of claim 12, wherein the vascular endothelial cell layer is usedas a carrier.
 17. The method of claim 12, wherein the extracellularmatrix consists of collagen, hyaluronic acid, fibronectin, vitronectin,and laminin.